Production of bypolymer film, fibre, foam and adhesive materials from soluble s-sulfonated keratin derivative

ABSTRACT

Film, fibre, foam and adhesive materials are produced from soluble S-sulfonated keratins. Once formed, the films, fibres, foams or adhesives are treated to modify the properties of the materials, in particular to improve the wet strength of the materials. Treatments used include removal of the S-sulfonate group by treatment with a reducing agent, treatment with an acid or treatment with a common protein crosslinking agent or treatment with a reduced form of keratin or keratin protein. The films are made by solvent casting a solution of S-sulfonated keratin proteins, the foam made by freeze-drying a solution of S-sulfonated keratin proteins and the fibres made by extruding a solution of a S-sulfonated keratin protein.

FIELD OF THE INVENTION

This invention relates to the preparation and use of soluble keratinderivatives in the production of a range of biopolymer materials such asfilms, fibres, foams and adhesives, and the improvement of thosematerials using further chemical treatments.

BACKGROUND TO THE INVENTION

Keratins are a class of structural proteins widely represented inbiological structures, especially in epithelial tissues of highervertebrates. Keratins may be divided into two major classes, the softkeratins (occurring in skin and a few other tissues) and hard keratins,forming the material of nails, claws, hair, horn and (in birds andreptiles) is feathers and scales.

The hard keratins may in turn be further subdivided into structuraltypes described as α-keratin, β-keratin, or feather keratin. Keratins ofthe α and β types have different predominant structural motifs in theirproteins, in the former case supramolecular structures based on theα-helix secondary structure of protein chains, and in the latter case onthe β-pleated sheet motif.

All keratins are characterised by a high level of the sulphur-containingdiamino-acid cystine, which acts as a cross-linking point betweenprotein chains. This feature of a high-level of interchain crosslinkingthrough cystine gives the keratins, especially the hard keratins, theircharacteristics of toughness, durability, resistance to degradation, anddesirable mechanical properties. Cystine contents vary widely in thekeratins, which is reflected in their variation in mechanicalproperties.

Wool and hair are examples of hard α-keratin. However, even in a givenα-keratin, there are many classes of structural protein present, and themechanical properties arise from a sophisticated supramolecularorganisation of proteins of many different types to create a complexmorphology with a correspondingly complex mechanical behaviour.

An object of the invention is to provide biopolymer materials derivedfrom soluble keratin derivatives and production methods for producingthe biopolymer materials.

SUMMARY OF THE INVENTION

According to a broadest aspect of the invention there are providedmaterials derived from S-sulfonated keratin proteins, as herein defined,in the form of films, fibres, foams or adhesives. The S-sulfonatedkeratin proteins can be derived from wool keratin and be enriched inintermediate filament protein(s).

According to another aspect of the invention there is provided a processmethod for the formation of films from S-sulfonated keratin proteins inwhich a solution of the proteins is cast and the solution solventsevaporated to leave a protein film.

The solution(s) used can be aqueous based, including some proportion oforganic solvents.

The films produced by this process method are inherently soluble inwater or the solvent mix used for casting the film.

Another aspect of the invention describes a method for improving the wetstrength of films, produced by the process method, by using chemicalagents, such as thiols and phosphines, that remove the sulfonate groupand allow the formation of disulfide bonds within the protein film. Thedifsulfide bonds provide the film with wet strength.

Another method of improving the wet strength of a film, produced by theprocess method, is described in which acidic solutions are used to treatthe protein film, and through a process of protonation of the sulfonategroups and any other suitable polar groups within the protein, the filmbecomes insoluble in water and has significant wet strength.

Another aspect of the invention describes introduction of crosslinksinto a film, produced by the process method, through the use ofcrosslinking agents such as those commonly used in proteinmodifications, that target a range of functional groups present withinthe protein.

A further aspect of the invention is a method for the production ofprotein films using a solution comprising a combination S-sulfonatedkeratin proteins and reduced keratin proteins or peptides containingreactive cysteine residues. The two species combine to form acrosslinked keratin network and subsequently a protein film with goodwet strength properties. This approach of combining S-sulfonated andreduced keratins can also be applied to the production of keratinfibres, foams and adhesives.

A further aspect of the invention is a method for the production ofkeratin fibres through the extrusion of a solution comprising ofS-sulfonated keratin proteins through a spinnerette into a coagulationbath that causes the protein to become insoluble. In particular thecoagulation bath may contain reductants, such as thiols or phosphines,that cause the removal of the sulfonate group from the protein and leadto disulfide groups forming. In addition the coagulation bath cancontain crosslinking agents, such as formaldehyde or glutaraldehyde,which cause the protein(s) to become insoluble on contact with thecoagulation bath. In addition the coagulation bath can be at acidic pH,which also causes the protein solution to become insoluble.

A further aspect of the invention is a method for the production ofkeratin fibres through the extrusion of a solution comprising ofS-sulfonated keratin proteins through a spinnerette into a hotenvironment through which the solvent is rapidly removed and a fibrouskeratin material remains. Fibres produced in this way can be furtherprocessed through wet chemical treatments to improve the wet strength ofthe fibres through the formation of crosslinks, or by protonation of theprotein in manners similar to those described above for keratin films.

A further aspect of the invention is a method for the production ofkeratin foams through the freeze drying of a solution of S-sulfonatedkeratin proteins. Foams produced in this way can be modified usingsimilar methods to those described for keratin films, that is throughthe use of a reductant such as a thiol or phosphine to remove theS-sulfonate group, through the use of reduced keratin proteins orpeptides to remove the S-sulfonate group, through the use of an acidicsolution to protonate the S-sulfonate group and the protein, or throughthe use of crosslinking agents such as formaldehyde and glutaraldehydeto modify the protein.

A further aspect of the Invention is a range of keratin based adhesives,comprising at least in part a solution of S-sulfonated keratin proteins.These adhesives can be made to have superior wet strength propertiesthrough the use of reducing agents, such as thiols or phosphines.Alternatively wet strength can be imparted through the use of a reducedkeratin protein or reduced keratin peptide, to create a crosslinkedkeratin network. These two sets of reagents can form a ‘two pot’adhesive.

The flexibility of the films, fibres, foams and adhesives produced bythe methods described can be modified through the use of plasticizerssuch as those from the glycerol or polyethylene glycol families.

According to further aspect of the invention there is provided a film,fibre, foam or adhesive material derived from keratin derivates of highmolecular weight as described and claimed in PCT/NZ02/00125 whereby theprocess includes a first stage digestion is step of sulfonating akeratin source by oxidative sulfitolysis followed by a second stagerepetitive aqueous extraction involving separation of soluble andinsoluble keratin and subsequent re-extraction of the insoluble keratinto thereby produce a highly S-sulfonated keratin derivative. The proteinkeratin source can be a naturally occurring protein source.

According to yet a further aspect of the invention there is provided afilm, fibre, foam or adhesive material derived from either highlyS-sulfonated keratin intermediate filament proteins, soluble keratinpeptides or a purified protein with little or no damage to thestructural integrity of the protein as produced from an impure proteinsource as described above.

According to yet a further aspect of the invention there is provided acombination of engineering solutions to produce a film, fibre, foam oradhesive material derived from S-sulfonated keratin proteins.

According to yet another aspect of the invention there is provided afilm, fibre, foam or adhesive material obtained from a protein producedfrom a large scale recovery method as described and claimed inPCT/NZ02/00125.

DESCRIPTION OF PREFERRED EXAMPLES

The features of this invention specifically cite some methods andapplications based on hard α-keratins from wool. However, the principlecan equally well apply to alternative α-keratins, or any source ofkeratin which is able to yield proteins of the intermediate filament(IF) type.

Similar preparative methods have been applied by the applicants to otherkeratin sources such as feathers, to produce materials equally wellsuited for some of the applications described below. The features ofthis invention are intended to cover the utilisation of such keratins aswell, in applications which are not dependent on the presence ofproteins of the α-type (IF proteins). This includes applications wherepreparations based on β or feather keratin may be combined with IFproteins.

The characteristics of toughness and insolubility typical of hardkeratins are desirable properties in many industrial materials. Inaddition, keratin materials are biodegradable and produced from asustainable resource and as such they have significant potential for useas a substitute for oil-based polymers in many applications, such asfilms, fibres and adhesives. Their use in cosmetics and personal careapplications is already well established and an extension to medicalmaterials is proposed using materials such as those outlined in thisspecification.

Wool represents a convenient source of hard α-keratins, although anyother animal fibre, or horns, or hooves, would serve equally well as asource of the desired proteins. Wool is composed of approximately 95%keratin, which can be broadly divided into three protein classes. Theintermediate filament proteins are typically of high molecular weight(45-60 kD), with a partly fibrillar tertiary structure and a cysteinecontent of the order of 6%. They account for approximately 58% of thewool fibre by mass although only part of this mass is actuallyhelix-forming in structure. The high- and ultra-high-sulphur proteins,approximately 26% of the wool fibre, are globular in structure, have amolecular weight range of 10-40 kD and can contain cysteine levels up to30 mol %. The high-glycine-tyrosine proteins are a minor classcomprising 6% of the wool fibre, have molecular weights of the order of10 kD and are characterised by their high content of glycine andtyrosine amino acid residues.

Proteins from the different classes of wool keratins possesscharacteristics that will give them unique advantages in specificapplications.

This invention pertains largely to the use of intermediate filamentproteins, and the use of them to produce films, fibres, foams andadhesives.

Nonetheless the other non-fibrillar proteins have applications in theirown right in more restricted fields.

Likewise feather keratins, derived by extractive procedures similar tothose applied to wool, have specific valuable applications in certainareas as defined below, but do not contain the IF proteins deemed to bedesirable in some end-uses.

The soluble keratin derivatives used In the method and subsequentchemical treatments described in this specification were obtained fromwool or feathers either by reduction using sodium sulphide or byoxidative sulphitolysis. An example of process for the production ofsoluble keratin derivates is described in the applicant's PCT/NZ02/00125patent specification, the description of which is incorporated herein byway of reference and outlined above. The reduction of wool or featherkeratin using sodium sulphide involves dissolution in a dilute sodiumsulphide solution (or other sulphide solution). The combination of highsolution pH and sulphide ion concentration results in the keratin beingdegraded to some extent, with possible hydrolysis of some of the peptidebonds occuring, as well as the disulphide bonds being reduced to yieldprotein rich in thiol and polysulphide functionality. The rich thiolfunction of the isolated protein can be confirmed using reagents such asnitroprusside. Oxidative sulfitolysis involves the conversion of thecysteine in keratin to S-sulfocysteine by the action of sodium sulphiteand an oxidant, No peptide hydrolysis occurs and the solubilised keratinhas a molecular weight distribution very similar to that in theunkeratinised state. Proteins derivatised in this way are referred toherein as S-sulfonated keratin proteins throughout the process methods,and are isolated from an oxidative sulfitolysis solution in the acidform, that is as kerateine S-sulfonic acid.

S-sulfonated keratin protein is soluble only as the salt, which can beprepared by the addition of base to the S-sulfonated keratin protein.For the preparation of Films from S-sulfonated wool keratin Intermediatefilament protein it is convenient to prepare a 5% protein solution bysuspending S-sulfonated keratin protein in water and adding base such assodium hydroxide or ammonia to give a final composition of 1 ml 1 MNaOH, or equivalent base, per gram of protein to a give a solution witha final pH In the range 9-10. Casting this solution onto a flat surface,such as a glass plate, and allowing the water and/or ammonia toevaporate at room temperature results in the formation of a keratinfilm. These keratin films have a high degree of clarity and have thephysical properties detailed in Table 1 below. In untreated films thereis likely to be little or no covalent bonding occurring between keratinproteins within the material as, the disulphide bonds present in theoriginal keratin have been converted to S-sulfocysteine. The hydrogenbonding and other non-covalent interactions occurring between theproteins are clearly significant, as the tensile strength of thematerial in the dry state is relatively high. The hydrogen bonding typeinteractions are overcome in the presence of water, reflected by thelarge decrease in tensile strength under wet conditions.

The physical properties of the materials derived from S-sulfonatedkeratin proteins depend to a large extent on the nature of theinteractions between the proteins comprising the material. These can beaffected significantly by a range of chemical treatments, with one ofthe most significant of these treatments being the use of a reductant toremove the sulfonate group from the protein to leave a thiol function.Under atmospheric conditions, or in the presence of an oxidant such asdilute hydrogen peroxide, these thiol functions recombine to formdisulfide bonds and return the chemical nature of the keratin materialto one much closer to the original form, that is proteins containing ahigh proportion of cystine disulfide links.

Treatment with a reducing agent, such as ammonium thioglycollate at pH 7for 30 minutes, or tributylphosphine for 24 hours, is an effective wayto remove the sulfonate function from S-sulfonated keratin. This can beconfirmed using infra-red studies as the S-sulfonate group gives rise toa strong, sharp absorbance at 1022 cm⁻¹ which is observed to disappearon exposure of the S-sulfonated to the reagents described.

In one aspect of the invention the reductant used to remove thesulfonate function and introduce cystine disulfides is itself a keratinprotein. Reduced keratin proteins, or keratin peptides, containing thethiol function can be readily produced by the process of sulphidedissolution described above. Keratin proteins prepared in this waycontain the cysteine reducing group which may covalently attach directlyto the S-sulfonate group to form a cystine disulfide. In this way acrosslinked keratin network is formed without the use of other agents.

In the case of S-sulfonated wool keratin intermediate filament proteinfilms reductive treatment significantly improves the wet strengthproperties of the material, as indicated by Table 1. The materialretains a good degree of flexibility when wet. Other chemical treatmentsalso affect the film properties. Treatment with an acid, such as 1Mhydrochloric acid, protonates the basic groups within the protein andconverts the S-sulfocysteine, present as the sodium or ammonium salt, toS-sulfonic acid. This can improve the hydrogen bonding interactions, asthe wet strength of the film clearly improves and no covalent bonds havebeen introduced. The S-sulfonate functionality, as determined byinfra-red absorption, remains intact. Standard protein crosslinkingtreatments, such as the use of formaldehyde or glutaraldehyde, alsoimprove the wet strength of the film, and introduce rigidity in both thewet and dry states. This is achieved through crosslinking the proteinsin a way that does not specifically target the sulfonate functionalityand many of the amino acid residues containing nucleophilic side groupssuch as lysine, tyrosine and cystine may be involved in crosslinking.

TABLE 1 Strength, extension and swelling data for protein films. Drystrength Wet strength % extension % extension Film and ×10⁻⁷ Nm⁻² ×10⁻⁷Nm⁻² at break dry at break wet treatment (cv) (cv) (cv) (cv) Untreated1.3 (11) 0.06 (15) 151 (24) 227 (20) Reductant 5.9 (7)  2.2 (21)  6 (16)208 (15) Acid 6.1 (3)  1.6 (14)  6 (31) 387 (6) Glutaraldehyde 5.0 (8) 1.9 (14)  4 (11)  4 (8) Formaldehyde 2.8 (16) 0.96 (8)  7 (41)  13 (25)cv = coefficient of variation, %, n = 5

Solutions of S-sulfonated keratin proteins can be used to producereconstituted keratin fibres by a variety of extrusion methods. Using awet spinning approach, similar in concept to the spinning of viscoserayon in which a solution of a material is extruded into a coagulationbath in which the material is insoluble, solutions of S-sulfonatedkeratin proteins can be extruded into solutions containing chemicalsthat make the protein become insoluble. Any of the three approachesdescribed for chemically treating S-sulfonated keratin films can beemployed in the coagulation bath used to generate keratin fibres. Byemploying reductants, such as ammonium thioglycollate, in thecoagulation bath, the S-sulfonated keratin proteins are converted backto keratins containing cystine disulfides through a wet spinningprocess, thereby producing reconstituted keratin fibres that have amultitude of disulfide links and good physical properties. By usingacidic conditions the S-sulfonated keratin proteins become protonatedand subsequently insoluble. By using crosslinking agents, such asformaldehyde or glutaraldehyde, the protein also becomes insoluble. Thecoagulation baths can also contain high concentrations of salt orsolvent to assist the process of fibre formation. In each caseprecipitation of the extruded protein occurs, possibly only in an outerskin of the extruded filament, and a fibre is formed with sufficientmechanical integrity to allow it to be collected from the coagulationbath and subjected to further treatments such as drawing or otherchemical processes.

A dry spinning approach can also be employed for the production ofreconstituted keratin fibres. The method is similar in concept to theformation of S-sulfonated keratin films described above, in whichsolvent is removed from an S-sulfonated keratin protein solution and akeratin material remains. In the formation of fibres this approach isemployed by extruding a solution of S-sulfonated keratin protein thathas a composition typically of 6-10% protein and up to 50% of a solventsuch as acetone, ethanol or isopropylalcohol, with the remaining portionof the solution being water and a base such as sodium hydroxide to givea pH of 9-10. This solution is extruded downwards into a chambercontaining a continuous downward hot air stream which causes the solventto rapidly evaporate, and an S-sulfonated keratin fibre remains.Subsequent chemical treatments, such as the reductive, acidic orcrosslinking treatments described for keratin films described above, canbe employed to impart wet strength properties to keratin fibres producedby this method.

Solutions of S-sulfonated keratins can be used to prepare highly porousprotein foams. This is achieved by freeze drying a solution, prepared asdescribed for the casting of keratin films. In order to produce foamsthe solution is cast onto an appropriate dish or surface and frozen,prior to being freeze dried. The resulting porous network is a foam ofS-sulfonated keratin protein. As with the film and fibre forms of thismaterial, applying chemical modifications to the protein has asignificant effect on the wet properties of the material. In particular,applying reductants such as ammonium thioglycollate or tributylphosphineunder similar conditions to those applied to the protein film, resultsin the removal of the S-sulfonate group and the formation of a networkof disulfide bonds, and subsequently decreases the solubility andincreases in the wet strength of the foam. A reduced form of keratin canalso be used to similar effect, again resulting in the formation of foamcomprising of a keratin protein interconnected through a network ofdisulfide bonds. Treatment of the foam with an acid, such as 1Mhydrochloric acid, results in protonation of any available groups withinthe material, such as the S-sulfonate group, and a subsequent increasein the wet strength of the material. Crosslinking agents, such asformaldehyde or glutaraldehyde, can also be used to significantly modifythe wet properties of the foam.

All the above applications relate preferentially to the case of IF-typeproteins prepared from hard α-keratins such as wool, but otherapplications such as the following one can use keratins from othersources, such as feather keratin.

Solutions of keratins obtained from wool or feathers by either reductionusing sodium sulphide or by oxidative sulphitolysis as described aboveshow significant adhesive properties in various applications. However,the wet strength of both of these adhesives is limited. Keratin madesoluble by sulphide reduction is degraded to some extent and containsprotein chains of lower molecular weight than in the original wool.S-sulfonate derived keratin polymers contain no covalent crosslinks andhydrogen bonding interactions are weakened significantly in water, asdemonstrated by the keratin films described above. However the wetstrength and adhesive properties can be greatly enhanced by reformingdisulphide cross-links, by adding an oxidant in the case ofsulphide-derived proteins, or a reducing agent in the case of theS-sulfonated keratin proteins. By such means very effective adhesivebonding can be achieved, for example in wood-particle composites bondedwith oxidised sulphide-derived proteins.

A particular feature of this invention relates to the recognition thatthe sulphide-derived protein and the S-sulfonated keratin proteins canbe used in conjunction to create highly cross-linked structures withvery superior properties. As noted above, the former class of proteincan be crosslinked by oxidation, and the latter by reduction. The twoprotein classes, one being in a reduced state and the other in anoxidised state, will when mixed form a self-crosslinking system. Ineffect, in such a system, an addition of sulphide-derived protein isacting as a reductant and crosslinking agent to convert the S-sulfonategroups in the other component to disulfide bonds.

Such a two-pot self-crosslinking system is a particular aspect of theinvention which will have applications in many forms of product, and hasthe advantage of eliminating volatile low molecular weight materials andthe necessity to use solvents in some forms of product fabrication. Thusit is to be expected that such composites can be formed from mixtures ofsolids or viscous dispersions without shrinkage.

In such two-component systems, the respective sulphide-derived andS-sulfonate keratin proteins can be produced from the same or differentkeratin sources. For example, if the mechanical property characteristicsassociated with IF proteins were desirable, the S-sulfonated keratinprotein could be derived from a hard α-keratin such as wool, and thesulphide-derived protein from another keratin source such as feathers.

An alternative two-component system is one which utilises a reductantfrom the thiol or phosphine family in addition to S-sulfonated keratinproteins. Combining solutions of these two materials results in theremoval the sulfonate group and formation of cystine disulfides in themanner described above for keratin films and fibres. This gives rise toan adhesive formulation with good wet strength properties.

By such means, proteins from sources other than hard keratins can beincorporated into many of the product classes described above, andtherefore the features in this invention encompass keratin sources ingeneral and are not restricted to hard α-keratins.

Polar, soluble reagents of low molecular weight, such as polyethyleneglycol or glycerol, can be employed as plasticising agents to givekeratin materials flexibility. These agents are best employed byinclusion in the keratin solutions used as the starting point for theformation of films, fibres or adhesives.

EXAMPLES Example 1a Preparation of a Keratin Film

In order to prepare an S-sulfonated keratin film, a 5% keratin proteinsolution was prepared by suspending 0.5 g S-sulfonated wool keratinintermediate filament protein in water, followed by the gradual additionof 0.5 ml of 1M sodium hydroxide to the vigorously stirred solution overapproximately 2 hours. The pH of the solution was carefully monitoredand observed to elevate to ˜pH10 upon immediate addition of base, andgradually fall as the base was absorbed by dissolution of the protein. Afinal pH of 9.5 was obtained. The protein solution was centrifuged at34,000 g to remove any insoluble material and the resulting solution wascast onto a 100 mm square petri dish and allowed to dry under ambientconditions. Following drying a clear protein film remained which couldbe easily removed from the petri dish.

Example 1b Disulfide Crosslinking of Protein Films

In order to improve the wet strength of S-sulfonated keratin films,disulfide crosslinks were introduced to the film by immersing the filmsproduced in Example 1a in a solution containing a reducing agent. Oneexample is a solution comprising 0.25M ammonium thioglycollate and 0.1 Mpotassium phosphate buffer adjusted to pH 7.0. Another example is asolution comprising 1M thioglycollic acid. Another example is a solutioncontaining 85 microlitres of tributyl phosphine in 20 ml of 10% (v/v)0.2M borate buffer in dimethyl formamide buffered to pH 9.0. Followingimmersion in the solution with gentle agitation for 30 minutes in thecase of the thiols and 24 hours in the case of the phosphine, thekeratin film was removed, rinsed briefly with water and allowed to dryunder ambient conditions.

Example 1c Protonation of Protein Films

In order to improve the wet strength of S-sulfonated keratin films, acidwas used to protonate all available sites on the proteins. This wasachieved through immersion of the film produced in Example 1a in 1Mhydrochloric acid for 30 minutes. Following a brief wash with water thefilm was allowed to dry under ambient conditions.

Example 1d Non-Disulfide Crosslinking of Protein Films

In order to improve the wet strength of S-sulfonated keratin filmscrosslinking agents were used to chemically bond proteins together. Inone case this was achieved through the use of a solution of 8%formaldehyde in 0.1M phosphate buffer at pH 7.0. The film was immersedin this solution for 30 minutes, washed briefly with water and allowedto dry under ambient conditions. In another case, crosslinking wasachieved through the use of a solution of 5% glutaraldehyde in 0.1Mphosphate buffer at pH7.0. The film was immersed in this solution for 30minutes, washed briefly with water and allowed to dry under ambientconditions for 30 minutes.

Example 1e Plasticising of Protein Films

In a variation of Example 1a, flexible protein films are made byincorporating glycerol or polyethylene glycol into the protein solutiondescribed in Example 1a at a level up to 0.2 g per g of protein prior tocasting the film. The resulting films have a greater flexibility, asdetermined by extension at break measurements, than the analogous filmscontaining no plasticiser.

Example 2a Production of Keratin Fibres through Wet Spinning andDisulfide Crosslinking

In order to prepare fibres derived from S-sulfonated keratin proteins aspinning dope was prepared in a similar manner to that prepared inExample 1a, with the difference being that for the extrusion of fibres,the concentration of protein in the solution was in the range 6-15%. Aplasticiser, such as those described in Example 1e, was added to thespinning dope. Following centrifuging to remove solids and entrained airthe dope was forced, using a positive displacement pump such as asyringe or gear pump, or air pressure, through a spinnerette into acoagulation bath. The coagulation bath had a composition of 1M ammoniumthioglycollate, 0.4M sodium phosphate, 0.25M sodium sulfate, 2% glycerolall set to pH 7.0.

Example 2b Production of Keratin Fibres Through Wet Spinning andNon-Disulfide Crosslinking

In a variation to Example 2a, fibres were extruded into a coagulationbath with a composition of 0.25M ammonium thioglycollate, 0.1M sodiumphosphate, 8% formaldehyde and 2% glycerol. This served to form toughfibres without forming disulfide bonds, as shown by infra red analysiswhich clearly indicated the presence of the S-sulfonate group.Subsequent treatment of the fibres with solutions containing reductants,such as ammonium thioglycollate at a concentration of 0.25M and a pH of7.0 with 0.1M potassium phosphate buffer, was sufficient to remove theS-sulfonate group and reform disulfide bonds.

Example 2c Production of Keratin Fibres Through Dry Spinning

In order to produce fibres through a dry spinning process, first aspinning dope was prepared in a similar manner to that described inExample 2a. In variation to the dope preparation a solvent such asacetone or isopropylacohol was added to the dope to give a finalcomposition protein in the range 6-15%, solvent in the range 20-50% andplasticiser in the range 1-3%. The dope was extruded through aspinnerette, using similar technology to that described in Example 2a,downwards into a chamber with a continuous downwards hot air stream.This caused the solvent to rapidly evaporate leaving a keratin fibre.Subsequent wet processing of the fibre, through the use of acid,reductant and crosslinking agents, of the type described in Examples 1,was used to improve the wet strength properties of the fibre.

Example 3a Production of a Keratin Foam

A solution of S-sulfonated keratin protein, prepared to a proteinconcentration of 5% as described in Example 1a, was used to create akeratin foam by freezing the solution in a 100 mm square petri dish andfreeze drying the resulting solid.

Example 3b Chemical Modification of Keratin Foam

Chemical solutions containing reductants, acids or crosslinking agents,of the described in Examples 1b, c, and d were applied to the keratinfoam, in a manner identical to that described for the keratin film. Akeratin foam with significantly reduced solubility and improved wetstrength resulted.

Example 4a Application of a Keratin Adhesive to Bind Wood

A solution of S-sulfonated keratin protein, prepared to a proteinconcentration of 5% as described in Example 1a, was used to bindwoodchips by mixing the keratin solution with woodchips in a ratio of 1ml solution per gram of woodchips. The mixture was then pressed andheated in a similar manner to the production of commercial ureaformaldehyde bound particle board (3 MPa, 180° C., 300 s), and a solidwood keratin composite resulted.

Example 4b Application of a Keratin Adhesive to Bind Textiles

A solution of S-sulfonated keratin protein, prepared to a proteinconcentration of 5% as described in Example 1a, was used to bind woollentextiles by coating one of the textile surfaces with the keratinsolution and pressing another textile onto the coated textile with theuse of a pinch roller system. Following the drying of the composition atelevated temperature a bonded textile was produced. In a smallvariation, plasticiser was included in the protein solution, in a mannersimilar to that described in example 1d, to produce a flexible adhesive.

Example 4c A Two Pot Adhesive System using a Reductant

An adhesive was made by combining a solution of S-sulfonated keratinprotein, prepared in the manner described in Example 1a, with a solutionof a reductant. The reductant solution contained 10%triscarboxyethylphosphine hydrochloride. When mixed in a ratio of 10parts keratin solution to 1 part reductant solution and applied to twowood surfaces this two pot formulation dried over 12 hours to create astrong bond between the wooden surfaces that remained strong in a moistenvironment. In a variation of this application a reductant solution wasused which contained 0.25M ammonium thioglycollate buffered to pH 7.0with 0.1M potassium phosphate. When mixed in a ratio of 10 parts keratinsolution to 1 part reductant solution and applied to two wood surfacesthis two pot formulation dried over 12 hours to create a strong bondbetween the wooden surfaces that remained strong in a moist environment.

Example 4d A Two Pot Adhesive System using Two Forms of keratin

An adhesive was made by combining a solution of S-sulfonated keratinprotein, prepared in the manner described in Example 1a, with a reducedkeratin peptide solution which contained sulphur amino acids primarilyin the form of cysteine and had a composition of 5% protein and 2%sodium sulphide. When mixed in equal parts and applied to two woodsurfaces this two pot formulation dried over 12 hours to create a strongbond between the wooden surfaces that remained strong in a moistenvironment. In a variation of this application, the reduced keratinpeptide was used in the form of a solid and mixed with the S-sulfonatedkeratin protein solution in a ratio of 5 parts S-sulfonated keratinsolution to 1 part reduced keratin solid and applied to two woodsurfaces this two pot formulation dried over 12 hours to create a strongbond between the wooden surfaces that remained strong in a moistenvironment.

Where in the description particular integers are mentioned it is to beappreciated that their equivalents can be substituted therefore as ifthey were set forth herein.

Thus by the invention there is provided a method for the preparation anduse of soluble keratin derivatives in the production of a range ofbiopolymer materials such as films, fibres, foams and adhesives, and theimprovement of those materials using further chemical treatment.

Particular examples of the invention have been described and it isenvisaged that improvements and modifications can take place withoutdeparting from the scope of the attached claims.

1-12. (canceled)
 13. A method for making protein fibres, the methodcomprising the step of extruding a solution of S-sulfonated keratinsinto an aqueous solution containing salts and a reductant that causesthe S-sulfonated keratins in solution to become insoluble.
 14. Themethod as claimed in claim 13 wherein the reductant is a thoil orthioglycollate salt.
 15. A method for making protein fibres, the methodcomprising the step of extruding a solution of S-sulfonated keratinsinto an aqueous solution containing salts, a reductant and acrosslinking agent that causes the S-sulfonated keratin in solution tobecome insoluble.
 16. The method as claimed in claim 15 wherein thereductant is thiol or thioglycollate salt.
 17. The method as claimed inclaim 15 wherein the crosslinking agent is formaldehyde.
 18. A methodfor making protein fibres, the method comprising the step of extruding asolution of S-sulfonated keratins into an aqueous solution containingsalts and an acid that causes the protein S-sulfonated keratins insolution to become insoluble.
 19. The method as claimed in claim 18wherein the acid is sulfuric acid.
 20. A method for making proteinfibres, the method comprising the step of extruding a solution ofS-sulfonated keratins in a solvent into a hot environment andevaporating away the solvent to leave a fibrous material behind. 21-41.(canceled)
 42. The method as claimed in claim 16 wherein thecrosslinking agent is formaldehyde.
 43. The method as claimed in claim20 further comprising the step of treating the fibrous material with areductant.
 44. The method as claimed in claim 43 wherein the reductantis a thiol or a phosphine.
 45. The method as claimed in claim 20 furthercomprising the step of treating the fibrous material with a reduced formof keratin or a reduced form of keratin peptide.
 46. The method asclaimed in claim 20 further comprising the step of treating the fibrousmaterial with an acid to protonate S-sulfonate groups within theS-sulfonated keratin protein and any other polar groups.
 47. The methodas claimed in claim 20 further comprising the step of treating thefibrous material with formaldehyde or glutaraldehyde.
 48. A fibreproduced by a method comprising the step of extruding a solution ofS-sulfonated keratins into an aqueous solution containing salts, areductant and a crosslinking agent that causes the S-sulfonated keratinin solution to become insoluble.
 49. The fibre as claimed in claim 48wherein the reductant is thiol or thioglycollate salt.
 50. The fibre asclaimed in claim 48 wherein the crosslinking agent is formaldehyde. 51.A fibre produced by a method comprising the step of extruding a solutionof S-sulfonated keratins into an aqueous solution containing salts andan acid that causes the S-sulfonated keratins in solution to becomeinsoluble.
 52. The fibre as claimed in claim 51 wherein the acid issulfuric acid.
 53. A fibre produced by a method comprising the step ofextruding a solution of S-sulfonated keratins in a solvent into a hotenvironment and evaporating away the solvent to leave a fibrous materialbehind.